Our studies of the cell invasion mechanism of Trypanosoma cntzi have led to a series of novel and unexpected findings. Prior to invasion, host cell lysosomes are recruited to the entry site and gradually fuse with the plasma membrane, as the parasites enter the cell. This unusual invasion process is mediated by parasite-induced signaling events involving transient elevations in the cytosolic CaZ*and cAMP of host cells. Elevation in cytosolic free Ca -[unreadable] is sufficient for triggering exocytosis of conventional lysosomes in many celt types, a process that we found to be regulated by Syt VII, a ubiquitously-expressed member of the synaptotagmin family of Ca'-* sensors. Syt VII was also found to regulate the repair of plasma membrane lesions, and the entry of T. cruzi trypomastigotes into host cells. Thus, our findings suggest that T. cruzi subverts the Syt VII-dependent, lysosome-mediated plasma membrane repair machinery as a strategy for cell invasion. We now plan to directly test this hypothesis, by taking advantage of several molecular tools developed in our laboratory to modulate mammalian cell functions involved in this process. In addition, we plan to extend our investigation of lysosome-mediated cell entry to the study of intracellular survival strategies that depend on lysosomal fusion. Our approach, as in previous years, will be to focus on recent advances in mammalian cell biology to study pathways involved in intracellular parasitism, and to explore unique aspects of host/parasite interactions to learn about mammalian cell function. Our specific aims are: 1) To analyze the effect of deficiency in the lysosomal synaptotagmin isoform Syt VII on the T. cruzi/host cell interaction. 2) To determine if there is a direct relationship between host cell wounding/repair and susceptibility to T. cruzi infection. 3) To identify specific host cell SNARES involved in lysosomal exocytosis and investigate their role on the T. cruzi cell entry process. 4) To investigate the role of Syt yenII in the intracellular fusion of lysosomes with phagosomes. This project will increase our understanding of the mechanism by which this important parasite invades cells, in addition to providing important new information on the role of lysosomes on intracellular parasitism.